Abstract Komagataella phaffii (Pichia pastoris) is a methylotrophic yeast that is favored by industry and academia mainly for expression of heterologous proteins. However, its full potential as a host for bio-production of valuable compounds cannot be fully exploited as genetic tools are lagging behind those that are available for baker's yeast. The emergence of CRISPR-Cas9 technology has significantly improved the efficiency of gene manipulations of K. phaffii, but improvements in gene-editing methods are desirable to further accelerate engineering of this yeast. In this study, we have developed a versatile vector-based CRISPR-Cas9 method and showed that it works efficiently at different genetic loci using linear DNA fragments with very short targeting sequences including single-stranded oligonucleotides. Notably, we performed site-specific point mutations and full gene deletions using short (90 nt) single-stranded oligonucleotides at very high efficiencies. Lastly, we present a strategy for transient inactivation of non-homologous end-joining (NHEJ) pathway, where KU70 gene is disrupted by a visual marker (uidA gene). This system enables precise CRISPR-Cas9 based editing (including multiplexing) and facilitates simple reversion to NHEJ-proficient genotype. In conclusion, the tools presented in this study can be applied for easy and efficient engineering of K. phaffii strains and are compatible with high-throughput automated workflows.

Tomas Strucko

Adrian-E Gadar-Lopez

Frederik B Frøhling

Emma T Frost

Esther F Iversen

Helen Olsson

Zofia D Jarczynska

Uffe H Mortensen

FEMS Yeast Research

FEMS YEAST RESEARCH 提供高质量原创研究文章、小型评论、致编辑的信函、观点和评论的高效出版，表达当前观点。该期刊将仅选择那些被认为与该领域具有重要相关性的手稿进行出版，并且通常不会考虑那些在很大程度上描述性而没有对潜在机制或生物学的见解的文章。欢迎提交任何酵母品种，只要它们报告的结果在下面概述的范围内并且对酵母领域具有重要意义。

FEMS Yeast Research is a high-quality academic journal that efficiently publishes original research articles, short reviews, letters to the editor, viewpoints, and commentaries. The journal only accepts manuscripts that are deemed to have significant relevance to the field of yeast research and typically does not consider articles that are largely descriptive without insights into potential mechanisms or biology. We welcome submissions on any yeast strains, provided that the reported results are within the scope outlined below and are of significant importance to the field of yeast research.

FEMS Yeast Research 是一本高质量的学术期刊，专注于原创研究文章、小型评论、致编辑的信函、观点和评论的高效出版。该期刊仅接受那些与酵母研究领域具有重要相关性的手稿，通常不考虑那些主要描述性且缺乏对潜在机制或生物学见解的文章。我们欢迎提交任何酵母品种的研究，只要其结果在本期刊的范围内，并对酵母领域具有重要意义。

Category	Quartile
生物学	4
生物学, 生物工程与应用微生物	4
生物学, 微生物学	4
生物学, 真菌学	4

Oligonucleotide-based CRISPR-Cas9 toolbox for efficient engineering of Komagataella phaffii

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